The broad aims of this research are to develop new protease inhibitors that can be used to investigate the molecular mechanisms underlying the pathway(s) of intracellular protein degradation in skeletal muscle. Protease inhibitors will be synthesize, characterized, and their effects on the degradation of proteins tested in muscle systems. Initially we have synthesized four stereoisomeric tripeptide analogs of pepstatin, characterized their inhibition of purified pepsin using two model substrates, and found all four analogs to be more effective than pepstatin at inhibiting intracellular proteolysis in human fibroblasts. The objective of this proposal are to synthesize peptides that are analogs and derivatives of the naturally occurring protease inhibitors, pepstatin, leupeptin, antipain, chymostatin, and elastatinal. These inhibitors will be tested in skeletal myoblasts in culture and in muscle preparation in vitro in order to select those compounds that are the most effective at delaying or blocking protein degradation. We then propose to test the most promising compounds as inhibitors of degradation in dystrophic muscle and, finally, as inhibitors of muscle degradation in dystrophic chickens and mice in vivo. In a parallel line of investigation, we plan to isolate proteases involved in muscle degradation, develop specific inhibitors of these enzymes, and utilize the inhibitors to study the role of each protease in in vivo protein degradation. Thus, we purpose to develop protease inhibitors for their use in studying the pathway(s) for protein degradation in higher organisms and for their evaluation as therapeutic agents in the prevention of muscle degradation resulting from muscular dystrophy and other wasting diseases.